This invention relates to crystalline N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-(6R)-pteridinyl)methyl]amino]benzoyl]-Lglutamic acid (termed crystalline or (6R)-tetrahydrofolic acid herein-below), to its use, and to a process for its preparation.
Tetrahydrofolic acid derivatives have 2 asymmetric centres. Since these derivatives are synthesized from folic acid, i.e. N-(pteroyl)-L-glutamic acid, the optically active C atom in the glutamic acid moiety is in the L form, while the optically active C atom in the 6-position, which is usually formed by hydrogenation of the double bond in the 5,6-position of the pteroyl radical, exists in the racemic, i.e. (6R,S) form. Accordingly, synthetic tetrahydrofolic acid derivatives are composed of a 1:1 mixture of 2 diastereomers.
Tetrahydrofolates are mainly used in the form of calcium 5-formyl-5,6,7,8-tetrahydrofolate (leucovorin) or calcium 5-methyl-5,6,7,8-tetrahydrofolate as pharmaceuticals for the treatment of megaloblastic folic acid anaemia, as an antidote for improving the tolerance of folic acid antagonists, specifically aminopterin and methotrexate in cancer therapy (xe2x80x9cantifolate rescuexe2x80x9d), for enhancing the therapeutic effect of fluorinated pyrdmidines and for the treatment of autoimmune diseases such as psoriasis and rheumatic arthritis, for improving the tolerance of certain antiparasitics, such as trimethoprim-sulfamethoxazole, and for reducing the toxicity of dideazatetrahydrofolates in chemotherapy. Tetrahydrofolic acid is also used as starting material for the preparation of a variety of tetrahydrofolic acid derivatives.
To date, the direct use of tetrahydrofolic acid as a pharmaceutically and as a starting material for the preparation of a variety of tetrahydrofolic acid derivatives was made impossible by the difficulty encountered when preparing tetrahydrofolic acid in a purity which is acceptable for a pharmaceutical active substance and by the extreme instability of tetrahydrofolic acid, in particular its pronounced sensitivity to oxidation [see, in this context, also A. L. Fitzhugh, Pteridines 4(4), 187-191 (1993)]. Various methods were developed to overcome this instability, and particular mention must be made in connection with the present invention of DE-OS 2 323 124. Specific mention must also be made of EP 600 460 in the context of processes for the preparation of tetrahydrofolic acid and in connection with the present invention. However, no process which is feasible on an industrial scale has been found to date for the preparation of ultrapure, sufficiently stable tetrahydrofolic acid which would allow the pharmaceutical application of tetrahydrofolic acid.
Surprisingly, it has been found that chemically and optically ultrapure (6S) or (6R) tetrahydrofolic acid with an outstanding stability can be obtained by crystallizing optically pure (6S)- or optically pure (6R)-, enriched (6S)- or enriched (6R)- or else (6R,S)-tetrahydrofolic acid. The resulting crystalline (6S)- and/or (6R)-tetrahydrofolic acid allows for the first time the use of the substance as a pharmaceutical or as a starting material for the industrial-scale preparation of other ultrapure tetrahydrofolic acid derivatives. (6S)-tetrahydrofolic acid is crystallized from a polar medium at a pH of xe2x89xa73.5, while (6R)-tetrahydrofolic acid is crystallized from a polar medium at a pH of xe2x89xa72.
Suitable polar media include but are not limited to: especially water or a mixture of water and an organic solvent which is miscible with water, such as water-soluble alcohols, for example methanol, ethanol, n-propanol, iso-propanol, ethylene glycol, a water-soluble lower aliphatic carboxylic acid, for example formic acid, acetic acid, lactic acid, or water-soluble amides, for example formamide, dimethylformamide, dimethylacetamide, 1-methylpyrrolidone, 2-methyl-pyrrolidone, 2-piperidinone. No particular restrictions apply to the nature of the solvent employed and the mixing ratio, since crystalline (6S)-tetrahydrofolic acid and crystalline (6R)-tetrahydrofolic acid in general have lower solubility characteristics than the corresponding amorphous forms.
To initiate crystallization of (6S)-tetrahydrofolic acid, a pH of between 3.5 and 6.5 is particularly suitable. To initiate crystallization of (6R)-tetrahydrofolic acid, a pH of between 2 and 5.5 is particularly suitable. The optimum pH for initiating crystallization depends on the materials employed and the intended object and can be determined by simple experiments. In general, a higher salt content in the starting solution will require a lower pH for initiating crystallization, and a lower pH for initiating crystallization requires a slower crystallization process since otherwise amorphous tetrahydrofolic acid precipitates at a pH of around 3. For example, direct crystallization of (6S)-tetrahydrofolic acid from a reaction solution obtained by reducing folic acid using borohydride strictly requires a pH of xe2x89xa64.8 for initiating crystallization. After crystallization has been initiated, the pH may be varied.
During the crystallization of (6S)-tetra-hydrofolic acid and also during the crystallization of (6R)-tetrahydrofolic acid, the pH rises or may be kept constant by adding an acid or buffer. In the case of the crystallization of (6S)-tetrahydrofolic acid, a pH of between 4.5 and 5.5 during the crystallization is preferred if it is intended to optically enrich (6S) tetrahydrofolic acid, while a pH of between 3.5 and 4.5 during the crystallization is preferred if it is intended to prepare stable crystalline (6S)-tetrahydrofolic acid. In the case of the crystallization of (6R)-tetrahydrofolic acid, a pH of between 3.5 and 4.5 during the crystallization is preferred independently of the desired result. The crystallization can be carried out in each case at room temperature, at elevated temperature or else at reduced temperature.
The time required for crystallization varies between a few minutes and several days. As a rule, longer crystallization times result in higher purity and more stable products.
(6S)- and (6R)-tetrahydrofolic acid crystallize spontaneously by slowly adjusting the pH, either starting from a pH which is lower than the pH suitable for initiating the crystallization of the isomer in question, or, preferably, starting from a higher pH. Crystallization may be triggered by seeding with the corresponding crystalline tetrahydrofolic acid in the pH range which is suitable for initiating crystallization of the isomer in question.
The starting material for the crystallization can be racemic (6R,S)-tetrahydrofolic acid, enriched (6S)- or (6R)-tetrahydrofolic acid as well as amorphous or crystalline (6S)- or (6R)-tetrahydrofolic acid. Suitable as starting material are not only isolated solid substances, such as, for example, (6R,S)-tetrahydrofolic acid, addition salts of sulphuric and sulphonic acid with (6S)-tetrahydrofolic acid prepared as described in EP 495 204, as well as tetrahydrofolic acid which has been prepared in situ from folic acid by catalytic hydrogenation or by reduction using boron hydride. (6R) tetrahydrofolic acid may be crystallized directly from the (6S)-tetrahydrofolic acid crystallization mother liquor. Both isomers may be crystallized either from a solution obtained, for example or by bringing the pH to  greater than 7 or  less than 2, or from a suspension.
By using amorphous or partially crystalline optically pure tetrahydrofolic acid or salts thereof as the starting material for the crystallization, the above described process yields crystalline tetrahydrofolic acid of previously unachieved purity ( greater than 98%) and, equally, previously unachieved stability.
The invention also relates to the use of crystalline (6S)- and/or (6R)-tetrahydrofolic acid as a component for the preparation of pharmaceuticals or for the preparation of other tetrahydrofolic acid derivatives since the quality of crystalline (6S)- and (6R)-tetrahydrofolic acid in solid form remains high over a virtually unlimited period due to its outstanding stability. The invention also relates to pharmaceutical preparations comprising crystalline (6S)- and/or (6R)-tetrahydrofolic acid. The pharmaceutical preparation is made by known processes, such as, for example, lyophilization. The uses and methods of use are analogous to those of known substances from the field of the tetrahydrofolates, such as, for example, 5-formyl-5,6,7,8-tetrahydrofolic acid.
The invention furthermore relates to a process for separating (6R,S)-tetrahydrofolic acid by fractional crystallization to give the two diastereomers (6S)- and (6R)-tetrahydrofolic acid. This process is very simple and high-yielding. Even upon the first crystallization of a crude racemic (6R,S)-tetrahydrofolic acid, crystalline (6S)-tetrahydrofolic acid is obtained in yields of above 70% while its (6S) component amounts to above 75%, and crystalline (6R)-tetrahydrofolic acid is obtained in yields of above 50% while its (6R) component amounts to above 80%. Further crystallization steps under analogous conditions allow crystalline (6S)- and (6R)-tetrahydro-folic acid with an isomeric purity of above 95% to be obtained.
(6R)- or (6S)-tetrahydrofolic acid can also be used directly without isolation for the preparation of other tetrahydrofolic acid derivatives. For example, enriched 5,10-methylene-(6S)-tetrahydrofolic acid can be prepared very easily by adding formaldehyde to a (6R)-tetrahydrofolic acid solution.
Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius and unless otherwise indicated, all parts and percentages are by weight.
The entire disclosures of all applications, patents and publications, cited above and below, and of corresponding Swiss application 01442/94-6, are hereby incorporated by reference.
The tetrahydrofolic acid contents and the isomer contents given in the following examples were in each case determined by HPLC. All tetrahydrofolic acid contents are based on the anhydrous substance.